Functional glutamate transporters are expressed in the carotid chemoreceptor.

BACKGROUND: The carotid body (CB) plays a critical role in cyclic intermittent hypoxia (CIH)-induced chemosensitivity; however, the underlying mechanism remains uncertain. We have demonstrated the presence of multiple inotropic glutamate receptors (iGluRs) in CB, and that CIH exposure alters the level of some iGluRs in CB. This result implicates glutamatergic signaling in the CB response to hypoxia. The glutamatergic neurotransmission is not only dependent on glutamate and glutamate receptors, but is also dependent on glutamate transporters, including vesicular glutamate transporters (VGluTs) and excitatory amino acid transporters (EAATs). Here, we have further assessed the expression and distribution of VGluTs and EAATs in human and rat CB and the effect of CIH exposure on glutamate transporters expression. METHODS: The mRNA of VGluTs and EAATs in the human CB were detected by RT-PCR. The protein expression of VGluTs and EAATs in the human and rat CB were detected by Western blot. The distribution of VGluT3, EAAT2 and EAAT3 were observed by immunohistochemistry staining and immunofluorescence staining. Male Sprague-Dawley (SD) rats were exposed to CIH (FIO2 10-21%, 3 min/3 min for 8 h per day) for 2 weeks. The unpaired Student's t-test was performed. RESULTS: Here, we report on the presence of mRNAs for VGluT1-3 and EAAT1-3 in human CB, which is consistent with our previous results in rat CB. The proteins of VGluT1 and 3, EAAT2 and 3, but not VGluT2 and EAAT1, were detected with diverse levels in human and rat CB. Immunostaining showed that VGluT3, the major type of VGluTs in CB, was co-localized with tyrosine hydroxylase (TH) in type I cells. EAAT2 and EAAT3 were distributed not only in type I cells, but also in glial fibrillary acidic protein (GFAP) positive type II cells. Moreover, we found that exposure of SD rats to CIH enhanced the protein level of EAAT3 as well as TH, but attenuated the levels of VGluT3 and EAAT2 in CB. CONCLUSIONS: Our study suggests that glutamate transporters are expressed in the CB, and that glutamate transporters may contribute to glutamatergic signaling-dependent carotid chemoreflex to CIH.

Chemotaxis cluster 1 proteins form cytoplasmic arrays in Vibrio cholerae and are stabilized by a double signaling domain receptor DosM.

Nearly all motile bacterial cells use a highly sensitive and adaptable sensory system to detect changes in nutrient concentrations in the environment and guide their movements toward attractants and away from repellents. The best-studied bacterial chemoreceptor arrays are membrane-bound. Many motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae's cytoplasmic chemoreceptor array and establish that it is formed by proteins from cluster I. We further identify a chemoreceptor with an unusual domain architecture, DosM, which is essential for formation of the cytoplasmic arrays. DosM contains two signaling domains and spans the two-layered cytoplasmic arrays. Finally, we present evidence suggesting that this type of receptor is important for the structural stability of the cytoplasmic array.

Isoflurane abolishes spontaneous firing of serotonin neurons and masks their pH/CO2 chemosensitivity.

Serotonin (5-hydroxytryptamine, 5-HT) neurons from the mouse and rat rostral medulla are stimulated by increased CO2 when studied in culture or brain slices. However, the response of 5-HT neurons has been variable when animals are exposed to hypercapnia in vivo. Here we examined whether halogenated inhalational anesthetics, which activate TWIK-related acid-sensitive K(+) (TASK) channels, could mask an effect of CO2 on 5-HT neurons. During in vivo plethysmography in mice, isoflurane (1%) markedly reduced the hypercapnic ventilatory response (HCVR) by 78-96% depending upon mouse strain and ambient temperature. In a perfused rat brain stem preparation, isoflurane (1%) reduced or silenced spontaneous firing of medullary 5-HT neurons in situ and abolished their responses to elevated perfusate Pco2. In dissociated cell cultures, isoflurane (1%) hyperpolarized 5-HT neurons by 6.52 ± 3.94 mV and inhibited spontaneous firing. A subsequent decrease in pH from 7.4 to 7.2 depolarized neurons by 4.07 ± 2.10 mV, but that was insufficient to reach threshold for firing. Depolarizing current restored baseline firing and the firing frequency response to acidosis, indicating that isoflurane did not block the underlying mechanisms mediating chemosensitivity. These results demonstrate that isoflurane masks 5-HT neuron chemosensitivity in vitro and in situ and markedly decreases the HCVR in vivo. The use of this class of anesthetic has a particularly potent inhibitory effect on chemosensitivity of 5-HT neurons.

Core unit of chemotaxis signaling complexes.

Bacterial chemoreceptors, histidine kinase CheA, and coupling protein CheW form clusters of chemotaxis signaling complexes. In signaling complexes kinase activity is enhanced several hundredfold and placed under receptor control. Activation is necessary to poise enzyme activity such that receptor control has physiologically relevant effects. Thus kinase activation can be considered the underlying core activity of signaling complexes. We defined the minimal physical unit that generates this activity using chemoreceptor Tar from Escherichia coli rendered water soluble by insertion into nanodiscs to (i) measure saturable binding of CheA and CheW to the smallest kinase-activating groups of receptor dimers and (ii) purify and characterize core units of signaling complexes. Purified complexes activated kinase almost as well as signaling complexes formed on arrays of receptors in isolated native membrane. Purified complexes contained two receptor trimers of dimers and two CheW for each CheA dimer, consistent with the approximately 1:1 CheACheW ratio determined by binding measurements. The 2:2:1 stoichiometry implied that CheA dimers, the enzymatically active form, connect two chemoreceptor trimers of dimers by interaction of one CheA protomer and a CheW with each trimer, an organization for which specific molecular interactions have previously been identified. The core unit associates six receptor dimers with a CheA dimer, providing sufficient capacity to account for much of the cooperativity and interdimer influence observed experimentally. We conclude that the 221 organization is the core structural and functional unit of chemotaxis signaling complexes and postulate that hexagonal arrays characteristic of signaling complexes are built from this unit.

A putative pheromone receptor gene expressed in human olfactory mucosa.

Pheromones elicit specific behavioural responses and physiological alterations in recipients of the same species. In mammals, these chemical signals are recognized within the nasal cavity by sensory neurons that express pheromone receptors. In rodents, these receptors are thought to be represented by two large multigene families, comprising the V1r and V2r genes, which encode seven-transmembrane proteins. Although pheromonal effects have been demonstrated in humans, V1R or V2R counterparts of the rodent genes have yet to be characterized.

Cross-linking evidence for motional constraints within chemoreceptor trimers of dimers.

Chemotactic behavior in bacteria relies on the sensing ability of large chemoreceptor clusters that are usually located at the cell pole. In Escherichia coli, chemoreceptors exhibit higher-order interactions within those clusters based on a trimer-of-dimers organization. This architecture is conserved in a variety of other bacteria and archaea, implying that receptors in many microorganisms form trimer-of-dimer signaling teams. To gain further insight into the assembly and dynamic behavior of receptor trimers of dimers, we used in vivo cross-linking targeted to cysteine residues at various positions that define six different levels along the cytoplasmic signaling domains of the aspartate and serine chemoreceptors, Tar and Tsr, respectively. We found that the cytoplasmic domains of these receptors are close to each other near the trimer contact region at the cytoplasmic tip and lie farther apart as the receptor dimers approach the cytoplasmic membrane. Tar and Tsr reporter sites within the same or closely adjacent levels readily formed mixed cross-links, whereas reporters located different distances from the tip did not. These findings indicate that there are no significant vertical displacements of one dimer with respect to the others within the trimer unit. Attractant stimuli had no discernible effect on the cross-linking efficiency of any of the reporters tested, but a strong osmotic stimulus reproducibly enhanced cross-linking at most of the reporter sites, indicating that individual dimers may move closer together under this condition.

Transmembrane signaling in chimeras of the Escherichia coli aspartate and serine chemotaxis receptors and bacterial class III adenylyl cyclases.

The Escherichia coli chemoreceptors for serine (Tsr) and aspartate (Tar) and several bacterial class III adenylyl cyclases (ACs) share a common molecular architecture; that is, a membrane anchor that is linked via a cytoplasmic HAMP domain to a C-terminal signal output unit. Functionality of both proteins requires homodimerization. The chemotaxis receptors are well characterized, whereas the typical hexahelical membrane anchor (6TM) of class III ACs, suggested to operate as a channel or transporter, has no known function beyond a membrane anchor. We joined the intramolecular networks of Tsr or Tar and two bacterial ACs, Rv3645 from Mycobacterium tuberculosis and CyaG from Arthrospira platensis, across their signal transmission sites, connecting the chemotaxis receptors via different HAMP domains to the catalytic AC domains. AC activity in the chimeras was inhibited by micromolar concentrations of l-serine or l-aspartate in vitro and in vivo. Single point mutations known to abolish ligand binding in Tar (R69E or T154I) or Tsr (R69E or T156K) abrogated AC regulation. Co-expression of mutant pairs, which functionally complement each other, restored regulation in vitro and in vivo. Taken together, these studies demonstrate chemotaxis receptor-mediated regulation of chimeric bacterial ACs and connect chemical sensing and AC regulation.

Role of HAMP domains in chemotaxis signaling by bacterial chemoreceptors.

Bacterial chemoreceptors undergo conformational changes in response to variations in the concentration of extracellular ligands. These changes in chemoreceptor structure initiate a series of signaling events that ultimately result in regulation of rotation of the flagellar motor. Here we have used cryo-electron tomography combined with 3D averaging to determine the in situ structure of chemoreceptor assemblies in Escherichia coli cells that have been engineered to overproduce the serine chemoreceptor Tsr. We demonstrate that chemoreceptors are organized as trimers of receptor dimers and display two distinct conformations that differ principally in arrangement of the HAMP domains within each trimer. Ligand binding and methylation alter the distribution of chemoreceptors between the two conformations, with serine binding favoring the "expanded" conformation and chemoreceptor methylation favoring the "compact" conformation. The distinct positions of chemoreceptor HAMP domains within the context of a trimeric unit are thus likely to represent important aspects of chemoreceptor structural changes relevant to chemotaxis signaling. Based on these results, we propose that the compact and expanded conformations represent the "kinase-on" and "kinase-off" states of chemoreceptor trimers, respectively.

The structural basis for signal promiscuity in a bacterial chemoreceptor.

Signalling through chemosensory pathways is typically initiated by the binding of signal molecules to the chemoreceptor ligand binding domain (LBD). The PcaY_PP chemoreceptor from Pseudomonas putida KT2440 is characterized by an unusually broad signal range, and minimal requisites for signal binding are the presence of a C6-membered ring and that of a carboxyl group. Previous studies have shown that only some of the multiple signals recognized by this chemoreceptor are of apparent metabolic value. We report here high-resolution structures of PcaY_PP-LBD in the absence and presence of four cognate chemoeffectors and glycerol. The domain formed a four-helix bundle (4HB), and both ligand binding sites of the dimer were occupied with the high-affinity ligands protocatechuate and quinate, whereas the lower-affinity ligands benzoate and salicylate were present in only one site. Ligand binding was verified by microcalorimetric titration of site-directed mutants revealing important roles of an arginine and number of polar residues that establish an extensive hydrogen bonding network with bound ligands. The comparison of the apo and holo structures did not provide evidence for this receptor employing a transmembrane signalling mechanism that involves piston-like shifts of the final helix. Instead, ligand binding caused rigid-body scissoring movements of both monomers of the dimer. Comparisons with the 4HB domains of the Tar and Tsr chemoreceptors revealed significant structural differences. Importantly, the ligand binding site in PcaY_PP-LBD is approximately 8 Å removed from that of the Tar and Tsr receptors. Data indicate a significant amount of structural and functional diversity among 4HB domains. DATABASES: The coordinates and structure factors have been deposited in the protein data band with the following IDs: 6S1A (apo form), 6S18 (bound glycerol), 6S33 (bound protocatechuate), 6S38 (bound quinate), 6S3B (bound benzoate) and 6S37 (bound salicylate).

Nutrient-sensing components of the mouse stomach and the gastric ghrelin cell.

BACKGROUND: The ability of the gut to detect nutrients is critical to the regulation of gut hormone secretion, food intake, and postprandial blood glucose control. Ingested nutrients are detected by specific gut chemosensors. However, knowledge of these chemosensors has primarily been derived from the intestine, while available information on gastric chemosensors is limited. This study aimed to investigate the nutrient-sensing repertoire of the mouse stomach with particular emphasis on ghrelin cells. METHODS: Quantitative RT-PCR was used to determine mRNA levels of nutrient chemosensors (protein: G protein-coupled receptor 93 [GPR93], calcium-sensing receptor [CaSR], metabotropic glutamate receptor type 4 [mGluR4]; fatty acids: CD36, FFAR2&4; sweet/umami taste: T1R3), taste transduction components (TRPM5, GNAT2&3), and ghrelin and ghrelin-processing enzymes (PC1/3, ghrelin O-acyltransferase [GOAT]) in the gastric corpus and antrum of adult male C57BL/6 mice. Immunohistochemistry was performed to assess protein expression of chemosensors (GPR93, T1R3, CD36, and FFAR4) and their co-localization with ghrelin. KEY RESULTS: Most nutrient chemosensors had higher mRNA levels in the antrum compared to the corpus, except for CD36, GNAT2, ghrelin, and GOAT. Similar regional distribution was observed at the protein level. At least 60% of ghrelin-positive cells expressed T1R3 and FFAR4, and over 80% expressed GPR93 and CD36. CONCLUSIONS AND INFERENCES: The cellular mechanisms for the detection of nutrients are expressed in a region-specific manner in the mouse stomach and gastric ghrelin cells. These gastric nutrient chemosensors may play a role modulating gastrointestinal responses, such as the inhibition of ghrelin secretion following food intake.

Activation of the retrotrapezoid nucleus by posterior hypothalamic stimulation.

The retrotrapezoid nucleus (RTN) contains chemically defined neurons (ccRTN neurons) that provide a pH-regulated excitatory drive to the central respiratory pattern generator. Here we test whether ccRTN neurons respond to stimulation of the perifornical hypothalamus (PeF), a region that regulates breathing during sleep, stress and exercise. PeF stimulation with gabazine increased blood pressure, phrenic nerve discharge (PND) and the firing rate of ccRTN neurons in isoflurane-anaesthetized rats. Gabazine produced an approximately parallel upward shift of the steady-state relationship between ccRTN neuron firing rate and end-tidal CO(2), and a similar shift of the relationship between PND and end-tidal CO(2). The central respiratory modulation of ccRTN neurons persisted after gabazine without a change in pattern. Morphine administration typically abolished PND and reduced the discharge rate of most ccRTN neurons (by 25% on average). After morphine administration, PeF stimulation activated the ccRTN neurons normally but PND activation and the central respiratory modulation of the ccRTN neurons were severely attenuated. In the same rat preparation, most (58%) ccRTN neurons expressed c-Fos after exposure to hypercapnic hyperoxia (6-7% end-tidal CO(2); 3.5 h; no hypothalamic stimulation) and 62% expressed c-Fos under hypocapnia (approximately 3% end-tidal CO(2)) after PeF stimulation. Under baseline conditions (approximately 3% end-tidal CO(2), hyperoxia, no PeF stimulation) few (11%) ccRTN neurons expressed c-Fos. In summary, most ccRTN neurons are excited by posterior hypothalamic stimulation while retaining their normal response to CNS acidification. ccRTN neurons probably contribute both to the chemical drive of breathing and to the feed-forward control of breathing associated with emotions and or locomotion.

Looking inside the box: bacterial transistor arrays.

One often compares cells to computers, and signalling proteins to transistors. Location and wiring of those molecular transistors is paramount in defining the function of the subcellular chips. The bacterial chemotactic sensing apparatus is a large, stable assembly consisting of thousands of receptors, signal transducing kinases and linking proteins, and is responsible for the motile response of the bacterium to environmental signals, whether chemical, mechanical, or thermal. Because of its rich functional repertoire despite its relative simplicity, this chemosome has attracted much attention from both experimentalists and theoreticians, and the bacterial chemotaxis response becoming a benchmark in Systems Biology. Structural and functional models of the chemotactic device have been developed, often based on particular assumptions regarding the topology of the receptor lattice. In this issue of Molecular Microbiology, Briegel et al. provide a detailed view of the receptor arrangement, unravelling the wiring of the molecular signal processors.

Location and architecture of the Caulobacter crescentus chemoreceptor array.

A new method for recording both fluorescence and cryo-EM images of small bacterial cells was developed and used to identify chemoreceptor arrays in cryotomograms of intact Caulobacter crescentus cells. We show that in wild-type cells preserved in a near-native state, the chemoreceptors are hexagonally packed with a lattice spacing of 12 nm, just a few tens of nanometers away from the flagellar motor that they control. The arrays were always found on the convex side of the cell, further demonstrating that Caulobacter cells maintain dorsal/ventral as well as anterior/posterior asymmetry. Placing the known crystal structure of a trimer of receptor dimers at each vertex of the lattice accounts well for the density and agrees with other constraints. Based on this model for the arrangement of receptors, there are between one and two thousand receptors per array.

The vomeronasal organ.

The vomeronasal organ (VNO) is a chemoreceptor organ enclosed in a cartilaginous capsule and separated from the main olfactory epithelium. The vomeronasal neurons have two distinct types of receptor that differ from each other and from the large family of odorant receptors. The VNO receptors are seven-transmembrane receptors coupled to GTP-binding protein, but appear to activate inositol 1,4,5-trisphosphate signaling as opposed to cyclic adenosine monophosphate. The nature of stimulus access suggests that the VNO responds to nonvolatile cues, leading to activation of the hypothalamus by way of the accessory olfactory bulb and amygdala. The areas of hypothalamus innervated regulate reproductive, defensive, and ingestive behavior as well as neuroendocrine secretion.

Requirement of the carboxyl terminus of a bacterial chemoreceptor for its targeted proteolysis.

The bacterium Caulobacter crescentus yields two different progeny at each cell division; a chemotactically competent swarmer cell and a sessile stalked cell. The chemotaxis proteins are synthesized in the predivisional cell and then partition only to the swarmer cell upon division. The chemoreceptors that were newly synthesized were located at the nascent swarmer pole of the predivisional cell, an indication that asymmetry was established prior to cell division. When the swarmer cell differentiated into a stalked cell, the chemoreceptor was specifically degraded by virtue of an amino acid sequence located at its carboxyl terminus. Thus, a temporally and spatially restricted proteolytic event was a component of this differentiation process.

Seven-transmembrane proteins as odorant and chemosensory receptors.

The olfactory systems of various species solve the challenging problem of general molecular recognition in widely differing ways. Despite this variety, the molecular receptors are invariably G protein-coupled seven-transmembrane proteins, and are encoded by the largest gene families known to exist in a given animal genome. Receptor gene families have been identified in vertebrates and two invertebrate species, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. The complexity of the odorant receptor repertoire is estimated in mouse and rat at 1000 genes, or 1 percent of the genome, surpassing that of the immunoglobulin and T cell receptor genes combined. Two distinct seven-transmembrane gene families may encode in rodents the chemosensory receptors of the vomeronasal organ, which is specialized in the detection of pheromones. Remarkably, these five receptor families have practically no sequence homology among them. Genetic manipulation experiments in mice imply that vertebrate odorant receptors may fulfill a dual role, also serving as address molecules that guide axons of olfactory sensory neurons to their precise target in the brain.

Chemoreceptors in Caulobacter crescentus: trimers of receptor dimers in a partially ordered hexagonally packed array.

Chemoreceptor arrays are macromolecular complexes that form extended assemblies primarily at the poles of bacterial cells and mediate chemotaxis signal transduction, ultimately controlling cellular motility. We have used cryo-electron tomography to determine the spatial distribution and molecular architecture of signaling molecules that comprise chemoreceptor arrays in wild-type Caulobacter crescentus cells. We demonstrate that chemoreceptors are organized as trimers of receptor dimers, forming partially ordered hexagonally packed arrays of signaling complexes in the cytoplasmic membrane. This novel organization at the threshold between order and disorder suggests how chemoreceptors and associated molecules are arranged in signaling assemblies to respond dynamically in the activation and adaptation steps of bacterial chemotaxis.

Physical responses of bacterial chemoreceptors.

Chemoreceptors of the bacterium Escherichia coli are thought to form trimers of homodimers that undergo conformational changes upon ligand binding and thereby signal a cytoplasmic kinase. We monitored the physical responses of trimers in living cells lacking other chemotaxis proteins by fluorescently tagging receptors and measuring changes in fluorescence anisotropy. These changes were traced to changes in energy transfer between fluorophores on different dimers of a trimer: attractants move these fluorophores farther apart, and repellents move them closer together. These measurements allowed us to define the responses of bare receptor oligomers to ligand binding and compare them to the corresponding response in kinase activity. Receptor responses could be fit by a simple "two-state" model in which receptor dimers are in either active or inactive conformations, from which energy bias and dissociation constants could be estimated. Comparison with responses in kinase-activity indicated that higher-order interactions are dominant in receptor clusters.

Is TrpM5 a reliable marker for chemosensory cells? Multiple types of microvillous cells in the main olfactory epithelium of mice.

BACKGROUND: In the past, ciliated receptor neurons, basal cells, and supporting cells were considered the principal components of the main olfactory epithelium. Several studies reported the presence of microvillous cells but their function is unknown. A recent report showed cells in the main olfactory epithelium that express the transient receptor potential channel TrpM5 claiming that these cells are chemosensory and that TrpM5 is an intrinsic signaling component of mammalian chemosensory organs. We asked whether the TrpM5-positive cells in the olfactory epithelium are microvillous and whether they belong to a chemosensory system, i.e. are olfactory neurons or trigeminally-innervated solitary chemosensory cells. RESULTS: We investigated the main olfactory epithelium of mice at the light and electron microscopic level and describe several subpopulations of microvillous cells. The ultrastructure of the microvillous cells reveals at least three morphologically different types two of which express the TrpM5 channel. None of these cells have an axon that projects to the olfactory bulb. Tests with a large panel of cell markers indicate that the TrpM5-positive cells are not sensory since they express neither neuronal markers nor are contacted by trigeminal nerve fibers. CONCLUSION: We conclude that TrpM5 is not a reliable marker for chemosensory cells. The TrpM5-positive cells of the olfactory epithelium are microvillous and may be chemoresponsive albeit not part of the sensory apparatus. Activity of these microvillous cells may however influence functionality of local elements of the olfactory system.

Spatial coding of enantiomers in the rat olfactory bulb.

Because of their unique properties, enantiomers (pairs of mirror-symmetric, nonsuperimposable molecules that differ only in optical activity and their interaction with other chiral molecules) have been instrumental in demonstrating that olfactory perception relies on molecular shape. To investigate how molecular structure is encoded by the olfactory system, we combined behavioral discrimination tasks with optical imaging of intrinsic signals. We found that rats can behaviorally discriminate members of a wide range of enantiomer pairs, and imaging revealed enantiomer-selective glomeruli in the olfactory bulb, indicating that the spatial pattern of glomerular activity provides sufficient information to discriminate molecular shape.